Human cytomegalovirus (HCMV) is one of the most common opportunistic infections in AIDS patients. My long-term research goal is to understand HCMV pathogenesis;specifically how HCMV interacts with host cells, replicates and causes disease. This proposal is designed to define the HCMV genes required for viral replication in vivo. Since animal models for studying HCMV replication and pathogenesis in vivo are not available, the severe combined immunodeficient mice (SCID) implanted with human fetal tissues provide us an alternative and valuable model for these studies. The HCMV clinical isolate Toledo strain replicates with high titers in the implanted human tissues in SCID mice;however, attenuated strain AD169 has completely lost this ability. The difference between these two strains is a 15-kb genomic segment, encoding 19 viral genes, that remains in all clinical isolates tested, including Toledo, but is deleted from the AD169 genome. We have demonstrated that the crucial genes required for HCMV in vivo replication are indeed localized in the region. In this proposal, a Toledo bacterial artificial chromosome (BAG) clone and a highly efficient recombination system will be used to generate deletion mutant viruses. This will significantly simplify and facilitate the construction of recombinant HCMV viruses. These recombinant viruses will be tested for their growth in the implanted human tissues in SCID mice and endothelial cells and macrophages. Finally, the viral genes needed for viral replication in these two systems will be mapped and analyzed. This work will allow us to identify genes essential for HCMV in vivo replication and pathogenesis. It should lead to a better understanding of the molecular basis of HCMV pathogenesis, and has the potential to help in the prevention and treatment of HCMV related diseases, especially in AIDS patients.